The goal of this proposal is to dissect the underlying mechanism by which enterotoxins cause Staphylococcus aureus infective endocarditis (IE). S. aureus present a significant clinical and public health problem, causing some of the most severe hospital- and community-associated illnesses and affecting approximately 500,000 individuals each year in the United States. S. aureus is the leading cause of IE in the developed world, affecting about 40,000 individuals per year in the U.S. and killing 20-66% of patients. S. aureus IE is also the most aggressive, tissue destructive, and lethal form of IE. Treatment of S. aureus IE is challenging, requiring prolonged antibiotic therapy or surgery to remove infected valves. Infections with methicillin-resistant S. aureus are frequent, complicate treatment, and increase mortality. Little is known about the S. aureus virulence factors critical for IE development and the mechanisms that lead to such an aggressive form of disease. The mechanistic understanding of IE is of utmost importance, given that its incidence, severity, and lethality have not been reduced in the last 50 years. Current evidence suggests that staphylococcal enterotoxin C (SEC), toxic shock syndrome toxin (TSST1), and the enterotoxin gene cluster (egc) play a novel and essential role in the etiology of IE caused by S. aureus. Enterotoxin deletion/complementation studies demonstrated the requirement of SEC, TSST1, and select egc toxins in development of S. aureus IE in a rabbit model of native valve IE. Staphylococcal enterotoxins are known for their potent superantigenic properties resulting in a CD4+ T cell dependent cytokine storm leading to inflammatory syndromes, toxic shock syndrome, or septic shock. While adaptive immune system activation is characteristic of staphylococcal enterotoxins, this is not their only biological function. Enterotoxins also directly interact with endothelial cells, epithelial cells, and adipocytes by a mechanism independent of superantigenic activity. In epithelial cells, activation is dependent on a dodecapeptide located at the base of the central a-helix of the molecule. In Aim 1, we will use strains expressing enterotoxins inactivated in ability to interact with the T cell receptor, MHC-II receptor, or endothelial cells and the rabbit model of IE to determine whether IE is due to superantigenic activity or dodecapeptide- mediated effects, or both. IE is an infection of the aortic endothelium. Infection and inflammation of the vascular endothelium are well-recognized mediators of vascular pathologies, such as atherosclerosis. Hence, Aim 2 will determine the mechanisms by which enterotoxins affect the endothelium to promote IE development. For this, we will use the rabbit model of IE, the rabbit aortic explant culture model, and the newly developed human aortic endothelial cell line to elucidate mechanisms in vivo, ex vivo, and in vitro. We expect our proposed studies to generate data that will significantly advance our understanding of S. aureus IE and provide insight of prognostic and therapeutic value to reduce IE severity and mortality.